Human primary immunodeficiency caused by hypomorphic mutation of the NF-?B essential modifier (NEMO) gene impairs several immunological functions and leads to grave infectious susceptibilities. The disease resulting from these mutations (NEMO-ID) arises from an inability of NEMO to effectively perform its typical function enabling activation of the NF-?B transcription factor after ligation of key immunoreceptors. Patients with NEMO-ID are vulnerable to infection with cytomegalovirus, which has led us to identify their having a defect of NK cell cytotoxicity. NK cells are lymphocytes of the innate immune system that are capable of killing through the process of directed secretion of lytic granules onto a target cell after forming an immunological synapse (IS). NK cells are especially useful in defense against cytomegalovirus and other viruses that employ strategies to evade adaptive immunity. This is underscored by several other primary immunodeficiencies affecting NK cells. Although a number of the immunological deficits in NEMO-ID are understood at a mechanistic level, that of NK cell cytotoxicity is not. Through the work proposed in this application we will determine the mechanism of the NK cell defect in NEMO-ID. This is relevant as NK cell deficiency adversely affects the health of patients with NEMO-ID and understanding its mechanism will likely suggest therapeutic strategies to specifically overcome it. Defining the mechanism of NK cell deficiency in NEMO-ID will also provide unexpected and novel insight into the function of cytolytic cells and the IS. In our studies of NK cells from NEMO-ID patients we have found that the defect is specific and can be circumvented after IL-2 stimulation. Thus it is unlikely that the NK cells are developmentally abnormal and a mechanism in which NEMO serves an acute role in NK cell cytotoxicity is likely. Through in vitro preliminary studies we have found that the NEMO-dependent NF-?B pathway is rapidly induced in NK cells after activation receptor ligation. This leads to rapid NF-?B-dependent gene transcription and synthesis of new proteins in a timeframe consistent with the cytolytic process. Using several approaches we have identified a limited number of NEMO- and NF-?B-dependent rapidly synthesized proteins in NK cells and are pursuing their role in enabling cytotoxicity and the lytic IS. Thus, our long term objective is to further establish this paradigm of NEMO function in NK cells and define a role for NEMO-dependent rapidly synthesized proteins in formation of the IS and cytotoxicity. Specifically we will: 1) define the stage of the NK cell IS that depends upon NEMO function and NF-?B activation;2) determine the role of rapidly synthesized NEMO-dependent proteins in NK cell function;3) determine if IL-2 can restore the expression of critical NEMO/NF-?B dependent proteins in NK cells with blocked NF-?B signaling. Cumulatively, these studies will determine the mechanism of NK cell deficiency in NEMO-ID, define a novel pathway of regulating cytolytic function and provide insight into potentially useful therapy for NEMO-ID patients. PUBLIC HEALTH RELEVANCE: Our proposal, "The Mechanism of NK Cell Defects in Human NEMO Deficiency", is aimed at understanding why patients with hypomorphic mutations in the NEMO gene have defective NK cell function. We believe the answer is that NEMO serves a surprising role in rapid transcription and protein synthesis to enable NK cell cytotoxicity. Our work will test this hypothesis and be of broad relevance in that it will define a novel mechanism by which immunological function can be regulated as well as more specific insight into NK cells in NEMO deficiency that may allow for rational therapies.